Recent Advances in the Modeling of Alzheimer's Disease

Abstract

Since 1995, more than 100 transgenic (Tg) mouse models of Alzheimer's disease (AD) have been generated in which mutant amyloid precursor protein (APP) or APP/presenilin 1 (PS1) cDNA is overexpressed ( 1st generation models ). Although many of these models successfully recapitulate major pathological hallmarks of the disease such as amyloid β peptide (Aβ) deposition and neuroinflammation, they have suffered from artificial phenotypes in the form of overproduced or mislocalized APP/PS1 and their functional fragments, as well as calpastatin deficiency-induced early lethality, calpain activation, neuronal cell death without tau pathology, endoplasmic reticulum stresses, and inflammasome involvement. Such artifacts bring two important uncertainties into play, these being (1) why the artifacts arise, and (2) how they affect the interpretation of experimental results. In addition, destruction of endogenous gene loci in some Tg lines by transgenes has been reported. To overcome these concerns, single App knock-in mouse models harboring the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (App^NL-G-F and App^NL-F mice) were developed ( 2nd generation models ). While these models are interesting given that they exhibit Aβ pathology, neuroinflammation, and cognitive impairment in an age-dependent manner, the model with the Artic mutation, which exhibits an extensive pathology as early as 6 months of age, is not suitable for investigating Aβ metabolism and clearance because the Aβ in this model is resistant to proteolytic degradation and is therefore prone to aggregation. Moreover, it cannot be used for preclinical immunotherapy studies owing to the discrete affinity it shows for anti-Aβ antibodies. The weakness of the latter model (without the Arctic mutation) is that the pathology may require up to 18 months before it becomes sufficiently apparent for experimental investigation. Nevertheless, this model was successfully applied to modulating Aβ pathology by genome editing, to revealing the differential roles of neprilysin and insulin-degrading enzyme in Aβ metabolism, and to identifying somatostatin receptor subtypes involved in Aβ degradation by neprilysin. In addition to discussing these issues, we also provide here a technical guide for the application of App knock-in mice to AD research. Subsequently, a new double knock-in line carrying the App^NL-F and Psen1 ^P117L/WT mutations was generated, the pathogenic effect of which was found to be synergistic. A characteristic of this 3rd generation model is that it exhibits more cored plaque pathology and neuroinflammation than the App^NL-G-F line, and thus is more suitable for preclinical studies of disease-modifying medications targeting Aβ. Furthermore, a derivative App^G-F line devoid of Swedish mutations which can be utilized for preclinical studies of β-secretase modifier(s) was recently created. In addition, we introduce a new model of cerebral amyloid angiopathy that may be useful for analyzing amyloid-related imaging abnormalities that can be caused by anti-Aβ immunotherapy. Use of the App knock-in mice also led to identification of the α-endosulfine-K _ATP channel pathway as components of the somatostatin-evoked physiological mechanisms that reduce Aβ deposition via the activation of neprilysin. Such advances have provided new insights for the prevention and treatment of preclinical AD. Because tau pathology plays an essential role in AD pathogenesis, knock-in mice with human tau wherein the entire murine Mapt gene has been humanized were generated. Using these mice, the carboxy-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON) was discovered as a mediator linking tau pathology to neurodegeneration and showed that tau humanization promoted pathological tau propagation. Finally, we describe and discuss the current status of mutant human tau knock-in mice and a non-human primate model of AD that we have successfully created.

Keywords: Alzheimer’s disease; amyloid – beta; amyloidosis; mouse model; non-human primate (NHP); somatostatin; tau propagation.

FIGURE 1

Mislocalization of APP in APP-overexpressing mice. App KO mice, WT mice, APP23 (APP-overexpressing mice) and App KI mice (App^{NL–F/NL–F}) were subjected to immunohistochemistry using antibodies to APP, 22C11 (upper panels) and synaptophysin, a synaptic vesicle marker (lower panels) as indicated. App KO mice were used as negative controls for APP staining. While APP is selectively expressed in the axons of WT and KI mice, APP23 expresses unphysiologically high levels of APP not only in the axons but also in the soma and dendrites. The scale bar indicates 2 mm.

FIGURE 2

Second generation mouse models of Alzheimer’s disease. See text for detailed explanation.

FIGURE 3

App^G–F mice suitable for studies of BACE1 inhibitors. The App^G–F line is devoid of the Swedish mutation that influences the β-secretase activity and elevates the quantity of CTFβ. (The App^G–F line instead carries a wild-type sequence: KM.) The App^G–F model would be appropriate for use in preclinical studies of β-secretase inhibitors without the interference of the Swedish mutation.

FIGURE 4

Scheme of App^NL–F × Psen1^P117L double-mutant mice. For the generation of the double-mutant mice, the App^NL–F line was crossbred with the Psen1^P117L line whose mutation was introduced in the endogenous Psen1 gene utilizing base editing technology. The synergistic effects of the pathogenic mutations in the App and Psen1 genes strongly accelerates the deposition of wild-type human Aβ in mouse brains.

FIGURE 5

AD pathology in the hippocampus of a 3rd generation model mouse. A 12-month-old App^NL–F X Psen1^P117L/WT mouse was analyzed by immunohistochemistry. Blue: Aβ plaques; red: microglia; green: astrocytes. The bar indicates 25 μm.

FIGURE 6

Outlined protocols for extraction and quantification of Aβ from tissues. See text for detailed explanation.

FIGURE 7

Reactivity of different antibodies to Arctic Aβ in App^NL–G–F mice. (A) Epitope map of anti-Aβ antibodies. (B,C) Quantification of Arctic Aβ species using BNT77 as a capture antibody. BNT77 binds to the mid-portion of Aβ [see epitope map (A)]. A sandwich ELISA kit (Wako, Japan) was used to quantify Aβx-40 (C) and Aβx-42 (D), respectively. (D,E) Quantification of Arctic Aβ species using BAN50 as a capture antibody. BAN50 binds to the N-terminal region of Aβ [see epitope map (A)]. A sandwich ELISA kit (Wako, Japan) was used to quantify Aβx-40 (D) and Aβx-42 (E), respectively. BNT77 and BAN50 captured Arctic Aβ more weakly than wild-type Aβ. (F) Immunohistochemistry using various anti-Aβ antibodies. Brain sections derived from App^NL–F mice (24 months old) were immunostained using antibodies with different epitopes after antigen retrieval as indicated (upper panels); those of App^NL–G–F mice (9 months old) were similarly immunostained (lower panels). Scale bars represent 500 μm.

FIGURE 8

Somatostatin receptor subtypes 1 and 4 (SST_1/4) regulate the Aβ-degrading enzyme NEP. The neuropeptide somatostatin (SRIF) was identified as a regulator of NEP activity through in vitro screening. Subsequent analysis of the effect of genetic deletion of somatostatin receptor (SST) subtypes in mice revealed that SST₁ and SST₄ regulate NEP in a redundant manner. This was further confirmed by concurrently deleting SST₁ and SST₄ in App KI mice, which aggravated the Aβ pathology. SST_1/4 can be either a combination of SST₁ and SST₄ homodimers or an SST₁/SST₄ heterodimer.

FIGURE 9

Role of ENSA in the regulation of NEP activity. Schematic illustration of the mechanism describing NEP activity in the brain. ENSA, a downstream protein of SST-SST_1/4 signaling, plays a role as a ligand of the K_ATP channel composed of sulfonylurea receptor subunit 1 (SUR1) and inwardly rectifying K⁺ channel 6.2 (Kir6.2), resulting in the activation of NEP. SST_1/4 can be either a combination of SST₁ and SST₄ homodimers or an SST₁/SST₄ heterodimer.

FIGURE 10

Propagation of AD-tau in mouse brains. Propagation of tau in each mouse line was observed 3 months after AD-tau injection. Brain sections were immunostained with AT8 (red). Humanization of the host animal tau affects the transmission of the pathogenic agents. App^NL–G–F/MAPT dKI mice exhibited greater pathological propagation than App^NL–G–F KI mice.

FIGURE 11

Functions of CAPON in neurodegeneration. (A) Brain sections from WT, P301S-Tau-Tg or nos1ap-/-/P301S-Tau-Tg mice stained by the conventional method using hematoxylin and eosin (H&E). A CAPON (Nos1ap) deficiency restores AD-related pathological phenotypes in P301S-Tau-Tg mice. (B) Scheme of CAPON action. Aβ pathology elevates the level and localization of CAPON in hippocampal pyramidal cells. CAPON-induced neuronal cell death is closely associated with the pathological tau protein, although there appears to be a tau-independent mechanism in play as well.

FIGURE 12

Photograph of common marmosets (Callithrix jacchus). The photo shows members of captive common marmoset family. Their small body size, fecundity, and high cognitive functions are a suitable model for neuroscience. The photograph of marmosets was taken by WK at CIEA.